1. Field
The invention relates to high-pressure fluid pumping systems, and more particularly to pump systems for HPLC and other chemical and biological analytical procedures.
2. State of the Art
Fluid pumping systems for high-pressure liquid chromatography (referred to hereinafter as HPLC) and the like are well known. In HPLC, a sample is applied to the top of a column which is packed with particles of a selected size and composition, and a solvent or solvent mixture is pumped through the column. Chemical components of the sample are eluted in the solvent from the lower end of the column at different times in a manner which reflects their chemical properties and composition. For reproducibility and high analytical accuracy, HPLC requires fluid pumping which is stable and essentially pulseless (smooth flow which does not vary during fill and pump strokes), with defined precise flow rates.
A common technique used to enhance separation of compounds by HPLC is to use two or more solvents and to vary the relative amounts of the solvents in the solvent mixture as it is being pumped through the column. This technique is often referred to as gradient separation or gradient HPLC. Formation of the gradient requires mixing of the two solvents in a controlled fashion prior to injecting the solvent mixture into the column. Typical prior art HPLC pumping systems use one of two main arrangements for mixing the solvents, as exemplified in U.S. Pat. Nos. 4,311,586 to Baldwin et al., and 4,714,545 to Bente et al. In both arrangements, the solvents are mixed together before entering the pump which pumps the mixture into the column.
A development of importance in the area of HPLC is the use of so-called "microbore" columns having an internal diameter (abbreviated herein as I.D.) of 1 millimeter or less. (See R. Scott and P. Kucera, J. Chromatogr. 169:51, 1979; F. Yang, J. Chromatogr. 236: 265, 1982; F. Yang, U.S. Pat. No. 4,483,733, (November 1984); D. Ishii et al., J. Chromatogr. 144: 157, 1977; D. Ishii et al., J. Chromatogr. 185: 73, 1979; T. Takeuchi et al., J. Chromatogr. 238: 409, 1982.) The advantages of microbore column HPLC over conventional HPLC include reductions of up to 100-fold each in the amounts of solvent and column packing required. Such reductions bring corresponding reduction not only in the initial cost of solvent and expensive column packing material, but in the amount of solvent which must be disposed of after use. Since many of the solvents used in HPLC have toxic components, the environmental benefit of microbore HPLC vs. conventional HPLC is substantial. Additionally, there are numerous advantages for various analytical procedures (see above references).
Instrumentation for micro-bore HPLC has been developed by several LC instrument manufacturers. The typical "1.0 mm. id. micro-HPLC pump" systems presently commercially available are modified versions of conventional low pressure proportioning HPLC gradient pumps (See H. Bente, et al. U.S. Pat. No. 4,714,545 (December 1987); G. Leka et al., U.S. Pat. No. 4,260,342, (April 1981); P. Trafford, U.S. Pat. No. 4,728,434 (March 1988); P. Achener, et al., U.S. Pat. No. 4,045,343 (August 1977); J. Rock, U.S. Pat. No. 4,128,476 (December 1978); H. Magnussen, Jr. U.S. Pat. No. 4,180,375 (December 1979); H. Magnussen, Jr., U.S. Pat. No. 4,131,393, (December 1978); R. Allington, U.S. Pat. No. 4,869,374 (September 1989). Such conventional systems use cam-driven pumps in which each solvent is drawn separately into the piston chamber by the fill stroke of the pump. Mixing occurs by turbulence during the fill stroke and/or by pumping the mixed fluids through a mixing unit before injecting it into the column. It is highly desirable to have the fill stroke extremely short in comparison to the pump stroke (U.S. Pat. No. 4,311,586 to Baldwin et al.). With cam-driven pumps, the desired ratio of the fill stroke to the total cycle is achieved by selecting the shape and dimensions of the cam.
However, it is difficult to dimensionally adapt such cam-driven pump designs to provide both low flow rates under high pressure and a very low fill stroke/stroke cycle ratio. As a practical matter, cam-driven pumps with the desired stroke ratios cannot be designed for flow rates lower than about 50 .mu.l per minute. Also, cams for these low flow rates are quite large, increasing the bulk of the pump which must be used within a relatively small area crowded with other apparatus.
Therefore, the modified conventional systems referred to in the preceding paragraph for microbore applications provide a lower flow rate to individual columns either by the split-flow technique (Sj. van der Wal et al., J. High Resolut Chromatogr. Commun. 6:216, 1983), or by reducing the volume of the piston chamber.
Unfortunately, such modified low pressure proportioning pump systems operate poorly at flow rates below 50 .mu.l/min in gradient HPLC with microbore columns. There are three major problem areas. First, the sum of the system volume including proportioning valves, piston chamber, inlet check valve and interface tubings is typically five to ten time greater than the amount of solvent eluted per minute, which places a lower limit on the minimum gradient step obtainable. For typical example, there may be a 100 .mu.l total system volume for a system operating at 10 .mu.l per minute. In this case, it takes about ten minutes for every gradient step change. Such a large minimum step provides very poor resolution for linear gradient elution.
Second, again because of the relatively large system volume, there is a long gradient delay time. Because the mixed solvent at the outlet cavity of the proportioning valves must travel through a piston chamber having a large liquid-end volume, in addition to the above-mentioned components, the effective gradient elution of the sample components in the column is delayed a long time. A typical pump liquid end volume of 2 ml therefore causes about 200 minutes gradient delay when operated at a column elution rate of 10 .mu.l/min.
The long delay times and the relatively large gradient steps are not only time-consuming for the user, but also allow significant diffusion of the solvents in the gradient to occur. As a result of such diffusion, the gradients are generally poorly reproducible and sample components are poorly separated (L. Snyder et al., "Reproducibility problems in gradient elution caused by differing equipment," LC-GC, Vol. 8, No. 7, p. 524, 1990).
A further disadvantage is that the gradient regeneration time is very long. A volume approximately three times that of the pump liquid end is required for purging and regeneration of the initial solvent composition. For the 2 ml liquid end volume of the previous example above, it will take 600 minutes to regenerate the initial solvent composition at a 10 .mu.l/minute elution rate.
One typical approach to alleviating these problems of cam-driven pumps at low flow rates is the split flow technique. The solvent gradient is generated at high flow rate to eliminate the problem of gradient delay. A microflow stream is then split at constant pressure from the main solvent stream and sent to the injector and column; the excess flow is usually discarded. Thus, the split-flow technique does not offer any reduction in solvent use over conventional methods. Also, because the gradient is split at constant pressure, the actual pressure in the microflow column has diminished stability and accuracy.
A further disadvantage of cam-driven pumps is that a single pump can only provide a limited range of flow rates. This is because different flow rate ranges require cams of substantially different size, and the position of the cam relative to the motor and the piston is determined by the cam dimensions. Changing the positions of the motor and piston to accommodate a cam of different size is impractical because of the sensitive alignment required in piston pumps.
An alternate approach for pumping in microbore column HPLC is the single-stroke syringe-type piston pump (M. Munk, U.S. Pat. No. 4,032,445 (June 1977), R. Brownlee, U.S. Pat. No. 4,347,131 (August 1982), R. Alligton, U.S. Pat. No. 4,775,481 (March 1988). This type of syringe pump is capable of delivering solvent at a few .mu.l/min. However, syringe-type pumps also have significant disadvantages for microbore gradient HPLC. First, it is difficult to maintain a constant flow rate during gradient elution, due to the continuously changing flow resistance. This variation in flow resistance is believed to be a consequence of solvent composition, solvent compressibility and syringe liquid volume changes. Second, it is necessary to refill the liquid phase in the syringe piston between each analysis to minimize solvent compressibility effect and ensure good flow rate reproducibility, but refilling of the syringe is generally slow. Third, for gradient elution multiple syringe pumps are required, and these are very costly.
Because of these and other disadvantages of available low flow rate pumping systems, the potential advantages of microbore HPLC have not been realized.
Consequently, a need remains for a simple and inexpensive pump which can provide pulseless, reproducible solvent flow under pressures of up to 10,000 psi at flow rates of 0.1 to 1200 .mu.l per minute or less. A need further remains for a pumping system having a greatly reduced liquid volume between the gradient mixing unit and the column injector, which can reproducibly provide gradient flows with small gradient steps and a short lead time.
A need also remains for a check valve which can provide fast and responsive action under very high pressures to reversals of flow pressure, and for mixing apparatus which does not cause produce particulates which can clog the flow stream at microflow rates. Still further, a need remains for a pump head check valve which is more durable under high pressures and does not need frequent replacement.